The invention relates to the use of S-ydcB and B-ydcB, which are essential bacterial genes useful for identifying antibacterial agents.
Bacterial infections may be cutaneous, subcutaneous, or systemic. Opportunistic bacterial infections proliferate, especially in patients afflicted with AIDS or other diseases that compromise the immune system. Most bacteria that are pathogenic to humans are gram positive bacteria. The bacterium Streptococcus pneumoniae, for example, typically infects the respiratory tract and can cause lobar pneumonia, as well as meningitis, sinusitis, and other infections.
The invention is based on the discovery that the S-ydcB gene in the gram positive bacterium Streptococcus pneumoniae and the B-ydcB gene in Bacillus subtilis are essential for survival. These genes are considered xe2x80x9cessentialxe2x80x9d genes, and the S-ydcB and B-ydcB polypeptides are considered xe2x80x9cessentialxe2x80x9d polypeptides. The invention features methods for using these genes and polypeptides to identify antibacterial compounds that inhibit a wide variety of bacteria (e.g., gram-positive and gram-negative bacteria, such as Streptococcus, Bacillus, and E. coli). Such inhibitors attenuate bacterial growth by inhibiting the activity of an S-ydcB or B-ydcB polypeptide (or homolog or ortholog thereof), or by inhibiting transcription of an S-ydcB or B-ydcB gene (or homolog or ortholog), or by inhibiting translation of the mRNA transcribed from an S-ydcB or B-ydcB gene (or homolog or ortholog). The S-ydcB and B-ydcB genes and polypeptides also are included within the invention and can be used in methods for identifying homologous genes and polypeptides in other bacterial strains.
The amino acid and nucleic acid sequences of S-ydcB are set forth in FIG. 1 as SEQ ID NOs:2 and 1, respectively. The amino acid and nucleic acid sequences of B-ydcB are set forth in FIG. 2 as SEQ ID NOs:4 and 3 respectively.
Now that the S-ydcB and B-ydcB genes described herein have been identified and shown to be essential for survival, these genes and polypeptides and their homologs can be used to identify antibacterial agents. xe2x80x9cHomologsxe2x80x9d are structurally similar genes contained within a species, while xe2x80x9corthologsxe2x80x9d are functionally equivalent genes in other species. The identified antibacterial agents can readily be identified with high throughput assays to detect inhibition of the S-ydcB or B-ydcB polypeptide, or essential polypeptides with which S-ydcB and B-ydcB associate (e.g., in a pathway). This inhibition can be caused by small molecules interacting with (e.g., binding directly or indirectly to) the S-ydcB or B-ydcB polypeptide or other essential polypeptides in that pathway.
In an exemplary assay, but not the only assay, a promoter that responds to depletion of the B-ydcB or S-ydcB polypeptide (or homolog thereof) by upregulation or downregulation is linked to. a reporter gene. To identify a promoter that is up- or down-regulated by the depletion of a B-ydcB or S-ydcB protein, the gene encoding the B-ydcB or S-ydcB protein is deleted from the genome and replaced with a version of the gene in which the sequence encoding the B-ydcB or S-ydcB protein is operably linked to a regulatable promoter. The cells containing this regulatable genetic construct are kept alive by the B-ydcB or S-ydcB polypeptide produced from the genetic construct containing the regulatable promoter. However, the regulatable promoter allows expression of the B-ydcB or S-ydcB polypeptide to be reduced to a level that causes growth inhibition. Total RNA prepared from bacteria under such growth-limiting conditions is compared with RNA from wild-type cells. Standard methods of transcriptional profiling can be used to identify mRNA species that are either more or less abundant (i.e., up- or down-regulated) when expressed under the limiting conditions. Genomic sequence information, e.g., from GenBank, can be used to identify the promoter that drives expression of the identified RNA species. Such promoters are up- or down-regulated by depletion of the B-ydcB or S-ydcB polypeptide.
Having identified a promoter(s) that is up- or down-regulated by depletion of a B-ydcB or S-ydcB polypeptide, the promoter(s) is operably linked to a reporter gene (e.g., xcex2-galactosidase, gus, or green fluorescent protein (GFP)). A bacterial strain containing this reporter gene construct is then exposed to test compounds. Compounds that inhibit the B-ydcB or S-ydcB polypeptide (or other polypeptides in an essential pathway in which the B-ydcB or S-ydcB polypeptide participates) will cause a functional depletion of the B-ydcB or S-ydcB polypeptide and therefore lead to an upregulation or downregulation of expression of the reporter gene. Because the polypeptides described herein are essential for the survival of bacteria, compounds that inhibit the B-ydcB or S-ydcB polypeptides in such an assay are expected to be antibacterial and can be further tested, if desired, in standard susceptibility assays.
Another suitable method for identifying antibacterial compounds involves screening for small molecules that specifically interact with (i.e., bind directly or indirectly to) the B-ydcB or S-ydcB polypeptide. A variety of suitable interaction and binding assays are known in the art as described, for example, in U.S. Pat. Nos. 5,585,277 and 5,679,582, incorporated herein by reference. For example, in various conventional assays, test compounds can be assayed for their ability to interact with a B-ydcB or S-ydcB polypeptide by measuring the ability of the small molecule to stabilize the B-ydcB or S-ydcB polypeptide in its folded, rather than unfolded, state. More specifically, one can measure the degree of protection from unfolding that is afforded by the test compound. Test compounds that bind the polypeptide with high affinity cause, for example, a large shift in the temperature at which the polypeptide is denatured. Test compounds that stabilize the B-ydcB or S-ydcB polypeptide in a folded state can be further tested for antibacterial activity in a standard susceptibility assay.
In a related method for identifying antibacterial compounds, the B-ydcB or S-ydcB polypeptide is used to isolate peptide or nucleic acid ligands that specifically bind the B-ydcB or S-ydcB polypeptide. These peptide or nucleic acid ligands are then used in a displacement screen to identify small molecules that interact with the B-ydcB or S-ydcB polypeptide. Such assays can be carried out essentially as described above.
Another suitable method for identifying inhibitors of the B-ydcB or S-ydcB polypeptide involves identifying a biochemical activity of the polypeptide and then screening for small molecule inhibitors of the activity using, for example, a high throughput screening method. S-ydcB and B-ydcB catalyze a reaction of CoenzymeA plus apo-Acyl Carrier Protein to produce holo-Acyl Carrier Protein and 3xe2x80x2,5xe2x80x2-ADP (PAP). Based on this activity, various biochemical assays can be set up as high throughput screens to detect compounds that inhibit the enzymatic activity of ydcB. For example, incorporation of a labelled :version of CoenzymeA into the holoACP protein can readily :be detected. The label can be fluorescent, radioactive, or any easily detectable moiety, such as biotin. The holo-ACP protein can be the Acyl Carrier Protein derived from; any of a wide variety of bacteria, or it can be a peptide fragment thereof or a fusion portein containing ACP sequences. In an alternative assay, the production of PAP from the catalytic reaction described above is detected. PAP can be detected in a calorimetric assay in which sulfotransferase uses PAP as a cofactor (Lin et al., Analytical Biochemistry).
The various B-ydcB and S-ydcB polypeptides can be used, separately or together, in assays to identify test compounds that interact with these polypeptides. Test compounds that interact with these polypeptides then can readily be tested, in conventional assays, for their ability to inhibit bacterial growth. Test compounds that interact with the B-ydcB or S-ydcB polypeptides are candidate antibacterial agents, in contrast to compounds that do not interact with the B-ydcB or S-ydcB polypeptides. As described herein, any of a variety of art-known methods can be used to assay for the interaction of test compounds with the B-ydcB and S-ydcB polypeptides.
The invention also includes a method for identifying an antibacterial agent where the method entails: (a) contacting an S-ydcB or B-ydcB polypeptide, or homolog thereof, with a test compound; (b) detecting binding of the test compound to the polypeptide or homolog; and, optionally, (c) determining whether a test compound that binds to the polypeptide or homolog inhibits growth of bacteria, relative to growth of bacteria cultured in the absence of the test compound that binds to the polypeptide or homolog, as an indication that the test compound is an antibacterial agent.
In still another method, interaction of a test compound with an S-ydcB or B-ydcB polypeptide (e.g., binding) can be detected in a conventional two-hybrid system for detecting protein/protein interactions (e.g., in yeast or mammalian cells). A test compound found to interact with the S-ydcB or B-ydcB polypeptide can be further tested for antibacterial activity in a conventional susceptibility assay. Generally, in such two-hybrid methods, (a) the S-ydcB or B-ydcB polypeptide is provided as a fusion protein that includes the S-ydcB or:B-ydcB polypeptide fused to (i) a transcription activation domain of a transcription factor or (ii) a DNA-binding domain of a transcription factor; (b) the test polypeptide is provided as a fusion protein that includes the test polypeptide fused to (i) a transcription activation domain of a transcription factor or (ii) a DNA-binding domain of a transcription factor; and (c) binding of the test polypeptide to the S-ydcB or B-ydcB polypeptide is detected as a reconstitution of a transcription factor. Homologs of the S-ydcB and B-ydcB polypeptides can be used in similar methods. Reconstitution of the transcription factor can be detected, for example, by detecting transcription of a gene that is operably linked to a DNA sequence bound by the DNA-binding domain of the reconstituted transcription factor (See, for example, White, 1996, Proc. Natl. Acad. Sci. 93:10001-10003 and references cited therein and Vidal et al., 1996, Proc. Natl. Acad. Sci. 93:10315-10320).
In an alternative method, an isolated nucleic acid molecule encoding an S-ydcB or B-ydcB polypeptide is used to identify a compound that decreases the expression of a B-ydcB or S-ydcB polypeptide in vivo. Such compounds can be used as antibacterial agents. To discover such compounds, cells that express an S-ydcB or B-ydcB polypeptide are cultured, exposed to a test compound (or a mixture of test compounds), and the level of expression or activity is compared with the level of S-ydcB or B-ydcB polypeptide expression or activity in cells that are otherwise identical but that have not been exposed to the test compound(s) Many standard quantitative assays of gene expression can be utilized in this aspect of the invention.
To identify compounds that modulate expression of an S-ydcB or B-ydcB polypeptide (or homologous sequence), the test compound(s) can be added at varying concentrations to the culture medium of cells that express a S-ydcB or B-ydcB polypeptide (or homolog), as described herein. Such test compounds can include small molecules (typically, non-protein, non-polysaccharide chemical entities), polypeptides, and nucleic acids. The expression of the S-ydcB or B-ydcB polypeptide is then measured, for example, by Northern blot PCR analysis or RNAse protection analyses using a nucleic acid molecule of the invention as a probe. The level of expression in the presence of the test molecule, compared with the level of expression in its absence, will indicate whether or not the test molecule alters the expression of the S-ydcB or B-ydcB polypeptide. Because the S-ydcB and B-ydcB polypeptides, and homologs thereof, are essential for survival, test compounds that inhibit the expression and/or function of the B-ydcB or S-ydcB polypeptide will inhibit growth of, or kill, the cells that express B-ydcB or S-ydcB polypeptides.
Typically, the test compound will be a small organic molecule. Alternatively, the test compound can be a test polypeptide (e.g., a polypeptide having a random or predetermined amino acid sequence; or a naturally-occurring or synthetic polypeptide) or a nucleic acid, such as a DNA or RNA molecule. The test compound can be a naturally-occurring compound or it can be synthetically produced, if desired. Synthetic libraries, chemical libraries, and the like can be screened to identify compounds that bind the S-ydcB or B-ydcB polypeptide. More generally, binding of test a compound to the S-ydcB or B-ydcB polypeptide or homolog can be detected either in vitro or in vivo. If desired, the above-described methods for identifying compounds that modulate the expression of the polypeptides of the invention can be combined with measuring the levels of the S-ydcB or B-ydcB polypeptides expressed in the cells, e.g., by performing a Western blot analysis using antibodies that bind an S-ydcB or B-ydcB polypeptide.
Regardless of the source of the test compound, the B-ydcB and S-ydcB polypeptides described herein can be used to identify compounds that inhibit the activity of an S-ydcB or B-ydcB protein or transcription of an S-ydcB or B-ydcB gene, or translation of the mRNA transcribed from such a gene. These antibacterial agents can be used to inhibit a wide spectrum of pathogenic or non-pathogenic bacterial strains, particularly gram-positive bacteria.
In other embodiments, the invention includes pharmaceutical formulations that include a pharmaceutically acceptable excipient and an antibacterial agent identified using the methods described herein. In particular, the invention includes pharmaceutical formulations that contain antibacterial agents that inhibit the growth of, or kill, pathogenic bacterial strains (e.g., pathogenic gram positive bacterial strains such as pathogenic Streptococcus strains). Such pharmaceutical formulations can be used in a method of treating a bacterial infection in an organism (e.g., a Streptococcus infection). Such a method entails administering to the organism a therapeutically effective amount of the pharmaceutical formulation, i.e., an amount sufficient to ameliorate signs and/or symptoms of the bacterial infection. In particular, such pharmaceutical formulations can be used to treat bacterial infections in mammals such as humans and domesticated mammals (e.g., cows, pigs, dogs, and cats), and in plants. The efficacy of such antibacterial agents in humans can be estimated in an animal model system well known to those of skill in the art (e.g., mouse and rabbit model systems of, for example, streptococcal pneumonia).
The invention further features methods of identifying from a large group of mutants those strains that have conditional lethal mutations. In general, the gene and corresponding gene product are subsequently identified, although the strains themselves can be used in screening or diagnostic assays. The mechanism(s) of action for the identified genes and gene products provide a rational basis for the design of antibacterial therapeutic agents. These antibacterial agents reduce the action of the gene product in a wild type strain, and therefore are useful in treating a subject with that type, or a similarly susceptible type, of infection by administering the agent to the subject in a pharmaceutically effective amount. Reduction in the action of the gene product includes competitive inhibition of the gene product for the active site of an enzyme or receptor; non-competitive inhibition; disrupting an intracellular cascade path which requires the gene product; binding to the gene product itself, before or after post-translational processing; and acting as a gene product mimetic, thereby down-regulating the activity.
Furthermore, the presence of the gene sequence in certain cells (e.g., a pathogenic bacterium of the same genus or similar species), and the absence or divergence of the sequence in host cells can be determined, if desired. Therapeutic agents directed toward genes or gene products that are not present in the host have several advantages, including fewer side effects, and lower overall dosage.
The invention also features an isolated S-ydcB polypeptide having the amino acid sequence set forth in SEQ ID NO:2, as depicted in FIG. 1, or conservative variations 15 thereof. An isolated nucleic acid encoding S-ydcB also is included within the invention. In addition, the invention includes (a) an isolated nucleic acid having the sequence of SEQ ID NO:1, as depicted in FIG. 1, or degenerate variants thereof; (b) an isolated nucleic acid having the sequence of SEQ ID NO:1, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and that hybridize under stringent conditions, as described below, to genomic DNA encoding the polypeptide of SEQ ID NO:2.
Identification of the S-ydcB gene allows homologs of the S-ydcB gene to be found in other strains within the species Streptococcus. The S-ydcB gene has an ortholog in Bacillus subtilis, termed xe2x80x9cB-ydcB,xe2x80x9d the amino acid and nucleic acid sequences of which are set forth in FIG. 2 (SEQ ID NOs:4 and 3,; respectively). The B-ydcB gene and polypeptide also can be used to identify compounds that inhibit bacterial growth (e.g., compounds that inhibit the activity of an S-ydcB or B-ydcB protein (or homolog or ortholog), or inhibit transcription of an S-ydcB or B-ydcB gene (or homolog or ortholog thereof)).
The S-ydcB polypeptides and genes described herein include the polypeptide and gene set forth in FIG. 1 herein, as well as isozymes, variants, and conservative variations of the sequences set forth in FIG. 1. The invention includes various isozymes of S-ydcB. For example, the invention includes a gene that encodes a S-ydcB polypeptide but which gene includes one or more point mutations, deletions, or promoter variants, provided that the resulting polypeptide retains a biological function of a S-ydcB polypeptide. The S-ydcB polypeptide has structural characteristics of acyl carrier protein synthase and displays such synthase activity in vitro. Thus, the various isozymes, variants, and conservative variations of the S-ydcB sequences set forth in FIG. 1 retain a biological function of S-ydcB as determined, for example, in an assay of acyl carrier synthase activity (e.g., as described above) or in a conventional complementation assay or binding assay. Also encompassed by the term S-ydcB gene are degenerate variants of the nucleic acid sequences set forth in FIG. 1 (SEQ ID NO:1). Degenerate variants of a nucleic acid sequence exist because of the degeneracy of the amino acid code; thus, those sequences that vary from the sequence represented by SEQ ID NO:1, but which nonetheless encode a polypeptide are included within the invention.
Likewise, because of the similarity in the structures of amino acids, conservative variations (as described herein) can be made in the amino acid sequence of the S-ydcB polypeptide while retaining the function of the polypeptide (e.g., as determined in a conventional complementation or binding assay). Other S-ydcB polypeptides and genes identified may be such conservative variations or degenerate variants of the particular S-ydcB polypeptide and nucleic acid set forth in FIG. 1 (SEQ ID Nos:1 and 2). Polypeptides that are substantially identical to the S-ydcB polypeptide and gene share at least 70%, e.g., 80% or 90%, sequence identity with SEQ ID Nos:2 and 1, respectively. Irrespective of the percent sequence identity between the S-ydcB sequence and the sequences represented by SEQ ID Nos:2 and 1, the S-ydcB genes and polypeptides encompassed by the invention preferably are able to complement for the lack of S-ydcB function (e.g., in a temperature-sensitive mutant) in a standard complementation assay. As described above for S-ydcB, the invention also includes various isozymes, variants, conservative variations and the like of the B. subtilis B-ydcB nucleic acid and polypeptide, which also has structural characteristics of acyl carrier protein synthases.
In various embodiments, the homologs or orthologs of the S-ydcB polypeptide used in the assays described herein is derived from a non-pathogenic or pathogenic gram positive bacterium.
The invention offers several advantages. For example, the methods for identifying antibacterial agents can be configured for high throughput screening of numerous candidate antibacterial agents. Because the B-ydcB and S-ydcB genes disclosed herein are thought to be highly conserved, antibacterial drugs targeted to these genes or their gene products are expected to have a broad spectrum of antibacterial activity.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the case of a conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative and are not intended to limit the scope of the invention, which is defined by the claims.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.